glycosylation and glycoproteins in thyroid cancer a potential role for diagnostics

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Glycosylation and Glycoproteins in ThyroidCancer: A Potential Role for Diagnostics

Anna Krzelak, Pawe Jwiak and Anna LipiskaUniversity of Lodz,

Poland

1. Introduction

Glycosylation is the most common and the most diverse form of co- and post-translationalmodifications. An analysis of the Swiss-PROT database has led to the estimation that morethan 50% of all proteins are glycosylated. Genes coding proteins involved in all types ofoligosaccharides biosynthesis represent 0.5 to 1% of the translated genome (Dennis et al.,1999). Glycoproteins are found ubiquitously in an organism either as soluble (intracellularor extracellular) or as membrane bound molecules. There is a great structural variety ofglycoproteins based on the type, length and linkage of a carbohydrate components as wellas the degree of saturation of potential glycosylation sites on the protein itself.

Carbohydrates can have a significant influence on the physicochemical properties ofglycoproteins, affecting their folding, solubility, aggregation and degradation. Furthermore,

glycan chains in glycoproteins play key roles in many biological processes such asembryonic development, immune response and cell-cell interactions in which sugar-sugaror sugar protein specific recognition is involved (Wei & Li , 2009).

Altered glycosylation is an universal feature of cancer cells, and certain glycan structures

and glycoproteins are well-known tumor markers. These include for example glycoproteinssuch as carcinoembryonic antigen (CEA), commonly used as a marker of colorectal cancer,prostate-specific antigen (PSA) and CA-125 used in the diagnosis of ovarian cancer (Drake etal., 2010). High expression of some glycosyl epitopes for example sialyl Lewis a, sialylLewis x, Lewis y, promotes invasion and metastasis. Antibodies against Lewis antigens areused for evaluation of specimens from breast, bladder, colorectal, esophageal and lung

carcinomas (Drake et al., 2010). Glycans can regulate different aspects of tumor progression,including proliferation, invasion and metastasis. Cancer-related changes in glycosylationcan reflect disease specific alterations in glycan biosynthetic pathways. These includevariations in the expression and activity of glycosyltransferases, enzymes that addmonosaccharides to acceptors, i.e. proteins or growing carbohydrate chains andglycosidases which catalyze the hydrolysis of the glycosidic linkage to release sugars.

There are three most common categories of protein glycosylation 1) N-glycosylation, whereglycans are attached to asparagine residues in a consensus sequence N-X-S/T viaN-acetylglucosamine (GlcNAc) residue; 2) O-glycosylation, where glycans are attached toserine or threonine viaN-acetylgalactosamine (GalNAc) residue (mucin type glycosylation);

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Updates in the Understanding and Management of Thyroid Cancer54

3) O-GlcNAcylation, where single N-acetylglucosamine residues are attached by O-linkageto serine and threonine residues (Fig.1). N-oligosaccharides have a common core structure offive sugars and differ in their outer branches. They are divided into three main classes: highmannose, complex and hybrid. High mannose oligosaccharides have additional mannoses

linked to the pentasaccharide core and forming the branches. Complex-typeoligosaccharides contain characteristic GlcNAc and Gal residues and are often terminatedwith sialic acid residues. Complex type oligosaccharides can be bi-, tri- or tetraantennary.

Fig. 1. Examples of carbohydrate structures. Arrows indicate the residues attached byglycosyltransferases the expression of which was studied in thyroid tumors:

GnT-V -N-acetylglucosaminyltransferase V , FUT8 1,6 fucosyltransferase,OGT O-GlcNAc transferase

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Glycosylation and Glycoproteins in Thyroid Cancer: A Potential Role for Diagnostics 55

Hybrid oligosaccharides contain one branch that has a complex structure and one or morehigh-mannose branches (Stanley et al., 2009). The mucin-type O-glycan, with N-acetylgalactosamine (GalNAc) at the reducing end, is a very common form of O-glycosylation in humans (Brockhausen, 1999; Wopereis et al., 2006). In O-glycosylated

proteins, oligosaccharides range in size from 1 to >20 sugars, displaying considerablestructural diversity. In total, 8 mucin-type core structures can be distinguished,dependingon the second sugar and its sugar linkage, of whichcores 16 and core 8 have been describedin humans. The N-acetylgalactosamine may be extended with sugars such as galactose, N-acetylglucosamine, fucose or sialic acid but not mannose, glucose or xylose residues(Brockhausen et al., 2009). The structural variability of glycans is dictated by tissue-specificregulation of glycosyltransferase genes, acceptor and sugar nucleotide donors availabilityand by competition between enzymes for acceptor intermediates during glycan elongation.Glycosyltransferases catalyze the transfer of a monosaccharide from specific sugarnucleotide donors onto particular position of a monosaccharide in a growing glycan chain in

one or two possible anomeric linkages (either or ) (Dennis et al., 1999). O-GlcNAcylationis a specific type of glycosylation since O-GlcNAc residues are not elongated and do notform complex structure. This dynamic and inducible modification is more similar tophosphorylation than to classical glycosylation (Hart & Akimoto, 2009).

2. Glycosylation in thyroid cancer

It is known that glycosylation profiles change significantly during oncogenesis (Wei & Li,2009). Detection of tumor specific alterations could be potentially useful for cancerdiagnostics. Lectins are a very good tool for the detection of changes in glycosylation. Theyare a group of proteins or glycoproteins that have affinity to carbohydrates and canreversibly and specifically interact with certain glycan structural motifs. Some lectins arewidely used in cancer diagnostics. For example Helix pomatia agglutinin (HPA) which

detects GalNAc1-4 Gal, is a part of a panel of markers for histological characterization of

gastric cancer specimens. HPA as well as Ulex europaeus agglutinin (UEA)(recognize Fuc1-

2Gal) is also used in breast cancer biopsy assessment (Drake et al., 2010).

It has been suggested that lectins might be useful histochemical tools for detecting andanalyzing poly-N-acetyllactosamines in thyroid normal and malignant tissues (Ito N et al.,1995,1996). Staining with lectins in combination with endo-beta-galactosidase digestiondemonstrated that poly-N-acetyllactosaminyl structures ubiquitously and consistentlyproduced in thyroid papillary carcinomas are highly heterogeneous in their chain lengthand branching status and quite different from those produced in other thyroid neoplasms(Ito N et al., 1995, 1996). Studies concerning glycosylation of intracellular proteins in benignand malignant thyroid neoplasms showed significant quantitative and qualitativedifferences in binding of Erythrina cristagalli (ECA) and Ricinus communis (RCA-120)agglutinins that recognize N-acetyllactosamine or galactose residues (Krzelak et al., 2003).In the majority of carcinoma samples lectin binding to cytosolic proteins was definitelyweaker in comparison with adenomas and non-neoplastic specimens which suggestsalterations in glycosylation of proteins in thyroid malignant tumors. Also binding patternsof Sambucus nigra (SNA) and Maakia amurensis (MAA)agglutinins which recognized sialicacids are different in malignant tumors compared with benign thyroid lesions (Krzelak etal., 2007).

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Changes in glycosylation are not the random consequence of disordered biology in tumorcells. It is characteristic that of all the possible glycan biosynthetic changes, only a limitedsubset of changes are correlated with malignant transformation (Varki et al., 2009).

2.1 N-acetylglucosaminyltransferase V

The most widely occurring glycosylation change in cancers is enhanced 1,6 GlcNAc side

chain branching (see Fig.1.). Increased amounts of 1,6 GlcNAc branched carbohydrateshave been linked to tumor invasion and metastasis in case of several human cancersincluding breast and colon. This carbohydrate structure is a product of increased activity ofN-acetylglucosaminyltransferase V (GnT-V) (Fernandes et al., 1991; Seelentag et al., 1998;Granovsky et al., 2000).

Ito Y et al. (2006) have studied by immunohistochemistry the expression of GnT-V inthyroid normal and cancer tissues. They found expression of this enzyme in 66 of 68 cases of

papillary carcinomas, 10 of 23 - follicular carcinomas, 9 of 13 - follicular adenomas and 6 of28 - anaplastic carcinomas but the enzyme was not expressed in normal thyroid tissue. It hasbeen shown that matriptase, a tumor-associated type II transmembrane serine protease is a

target protein for GnT-V and 1,6 GlcNAc branching of oligosaccharide on matriptaseincreases resistance of this protein to proteolytic degradation. Increased expression ofmatriptase and GnT-V levels were characteristic for papillary carcinoma. Althoughmatriptase is proposed to modulate the metastatic potential of some cancer cells, it is notlikely that the matriptase and GnT-V promote invasion and metastasis of papillary thyroidcancers. The levels of expression of both matriptase and GnT-V were significantly high inmicrocarcinomas. In case of poorly differentaiated and undifferentiated (anaplastic)carcinomas which show local and distant metastasis, expression of these proteins

significantly decreased. Thus matriptase and GnT-V probably play a role in early phases ofpapillary carcinomas growth, but not in their progression (Miyoshi et al., 2010).

2.2 Changes in sialic acid expression

Residues of sialic acid are usually found at the non-reducing terminal position of

glycoconjugate sugar chains, 2,3- or 2,6-linked to a galactose (Gal), or 2,6-linked to aN-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc) residues (Harduin-Lepers et al., 2001). Changes in sialic acid expression may be important in cancerprogression and metastasis for several reasons. First, sialic acid can prevent cell-cellinteractions through charge repulsion effects; second, sialylated structures can be

recognized by cell adhesion molecules belonging to selectin or siglec families; third, theaddition of sialic acid may mask the underlying sugar structure, thus avoiding recognitionby other lectins such as galectins (DallOlio & Chiricolo, 2001).

Nozawa et al. (1999) investigated immunohistochemically the reactivity of monoclonalantibody of FB21, which recognizes a sialic acid-dependent carbohydrate epitope withthyroid lesions in order to evaluated the potential diagnostic usefulness of sialic acidexpression analysis. The increased FB21 antigen reactivity appeared to be characteristic forfollicular and papillary thyroid tumors but not for medullary thyroid carcinoma. FB21reacted with almost all cases of follicular carcinomas and less than half of cases of papillarycarcinomas. A positive reaction was found on the cell surface membranes or apical parts of

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neoplastic follicles. The normal thyroid follicles, goiters, medullary and anaplasticcarcinomas were negative for FB21 reactivity. High frequency of reactivity of follicularcarcinoma with FB21 suggested that it may be useful in diagnosis of this histological type ofthyroid tumors.

Differences in sialic acid level on luminal surface between thyroid carcinomas and normalthyroid tissue or adenomas and goiters was observed in histochemistry studies using sialicacid binding lectins Tritrichomonas mobilensis lectin (TML), Maackia amurensis agglutinin(MAA) and Sambucus nigra agglutinin (SNA) (Babl et al., 2006). Malignant tumorsespecially papillary carcinoma had an increased level of sialic acid mainly alpha-2,3-linkedand recognized by MAA. These results suggest that increased membrane sialic acid onthyroid gland cells may be an important diagnostic indicator, that could be useful in thedistinction of malignant from benign thyroid lesions (Babl et al., 2006).

Cellular sialic acid level is mainly controlled metabolically by sialyltransferases andsialidases. Human sialyltransferases are a family of at least 18 different members that

catalyse the transfer of sialic acid residues from their activated form CMP-sialic acid toterminal position of oligosaccharide chains (Harduin-Lepers et al., 2001). Changes insialyltransferases expression have been observed in cancer tissues and the regulation of theirexpression is achieved mainly at the transcriptional level (DallOlio & Chiricolo, 2001).Sialidases catalyse the removal of sialic acid residues which is an initial step in thedegradation of glycoproteins. There are only few studies focusing on sialic acid metabolism

in thyroid tissue. Thyroid sialyltransferase 1 (-galactoside -2,6-sialyltransferase) and 4 (-galactoside -2,3-sialyltransferase) mRNA level and activity are increased in Graves disease(Kiljaski et al., 2005). Sialyltransferase 1 showed also a very evident increase of expressionin autonomously functioning thyroid nodules (AFTNs) versus their surrounding tissues(Eszlinger et al., 2004). Since sialyl Lewis epitopes are more frequently expressed inpapillary carcinomas than Lewis a and Lewis b it is speculated that a specific

glycosyltransferase, i.e. 2,3 sialyltransferase may be highly activated as compared with1,4 fucosyltransferase. Moreover, this sialyltransferase seems to be more strongly enhancedin papillary carcinoma than in follicular carcinoma (Kamoshida et al., 2000).

2.3 Fucosylation

Fucosylated glycans are synthesized by fucosyltransferases. Thirteen fucosyltransferasegenes have thus far been identified in the human genome. Based on the site of fucose

addition fucosyltransferases are classified into1,2 (FUT1, FUT2), 1,3/4 (FUT 3, 4, 5, 6, 7,

9), 1,6 (FUT8) and O-fucosyltransferase (POFUT1) groups (Becker & Lowe, 2003; Ma et al.,2006). Two additional 1,3 fucosyltransferase genes, FUT10 and FUT11 and oneO-fucosyltransferase gene have been identified in the human genome but their proteinproducts have not yet been demonstrated to be enzymatically active although they shareprimary sequence similarity with active fucosyltransferases. Certain types of fucosylatedglycoproteins for example alfa-fetoprotein and several kinds of antibodies, which recognizefucosylated oligosaccharides such as sialyl-Lewis a/x, have been used as tumor markers(Miyoshi et al., 2008). The Lewis blood group antigens are a related set of glycans that carry

1,2/1,4 fucose residues (Fig.2). Although growing evidence supports the functionalsignificance of fucosylation at various pathophysiological steps of carcinogenesis and tumorprogression, the significance of this modification in thyroid cancer has not been widely

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explored. The results of studies of Vierbuchen et al. (1992) concerning blood group antigensin medullary thyroid carcinoma (MTC) and normal thyroid tissue supported the generalconcept demonstrated in other carcinomas, that fucosyl- and sialyltransferases might bepreferentially activated. The Lewis antigens were absent in normal tissue and present in

MTC. Miettinen and Krkkinen (1996) analysed immunohistochemically a wide range ofthyroid tumors and non-neoplastic tissues by using CD15 antibody, which recognized Lewis xblood group antigen. Nodular goiter and papillary hyperplasia cases either showed noreactivity or were focally positive. Most papillary carcinomas were CD15 positive usually inthe majority of tumor cells. However only 50% of follicular carcinomas were positive andanaplastic carcinomas were negative. Similar results were obtained by Fonseca et al. (1996,1997) who found by using SH1 antibodies that most of papillary and follicular carcinomaswere extensively immunoreactive for Lewis x antigen in contrast to the absence ofexpression of this antigen in normal thyroid.

Fig. 2. Lewis antigens.

Ito Y et al. (2003) performed immunohistochemical studies of FUT8 expression in thyroidcancers. FUT8 catalyses the transfer of a fucose residue to the C6 position of the innermostGlcNAc residue of N-linked oligosaccharides on glycoproteins to produce core fucosylation(Fig.1.). The expression of FUT8 was very low in normal follicules. A high expression ofFUT8 was observed in 33.3% of papillary carcinomas and the incidence was directly linkedto tumor size and lymph node metastasis. But the number of cases of follicular carcinomaswith high expression of FUT8 was rather low. These results suggest that FUT8 expressionmay be a key factor in the progression of thyroid papillary carcinomas, but not follicular

carcinomas ( Ito Y et al., 2003; Miyoshi et al., 2010).

2.4 O-GlcNAcylation

O-GlcNAcylation is a post-translational protein modification consisting ofN-acetylglucosamine moiety attached by O-glycosidic linkage to serine and threonineresidues (Hart et al., 2007). O-GlcNAc is a unique type of glycosylation since it is notelongated to more complex glycan structures and is nearly exclusively on cytoplasmic andnuclear proteins. Furthermore, the addition and removal of O-GlcNAc moieties cycles in avery dynamic and inducible manner in response to different stimuli such as hormones,growth factors, and mitogens. That makes O-GlcNAcylation more similar to

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phosphorylation than to classical glycosylation. O-GlcNAcylation is one of the mostcommon post-translational modifications. So far, nearly one thousand of cellular proteinshave been identified to be O-GlcNAcylated (Butkinaree et al., 2010). These proteins belongto diverse functional groups and include nuclear pore proteins, transcription factors, RNA

binding proteins, cytoskeletal proteins, chaperones, phosphatases, kinases and otherenzymes (Zachara & Hart, 2006). There is growing evidence that O-GlcNAc modification isinvolved in a wide range of biological processes, such as signal transduction, transcription,cell cycle progression and metabolism (Hart et al., 2007). O-GlcNAc can modulate proteinfunction by regulating protein activity, protein-protein interaction, localization and proteindegradation (Zeidan & Hart, 2010).

There is a relationship between O-GlcNAcylation and phosphorylation. All O-GlcNAc

modified proteins are also phosphoproteins and sometimes O-GlcNAc and O-phosphate

moieties can compete for a binding site or alternatively O-GlcNAcylation and

phosphorylation can compete via steric hindrance when the substrate modification sites are

within several aminoacids from each other (Hu et al., 2010). However, unlikephosphorylation, where many kinases and phosphatases regulate the addition and removal

of phosphate, O-GlcNAcylation is regulated only by two enzymes. The addition of

O-GlcNAc to proteins is catalyzed by O-GlcNAc transferase (OGT) and its removal is

catalyzed by O-GlcNAc- selective N-acetyl--D-glucosaminidase (O-GlcNAcase, OGA) (Iyer

& Hart, 2003). OGT is encoded by a single gene on X chromosome (Lubas & Hanover, 2000;

Shafi et al., 2000). Although there is only one OGT gene, human OGT has at least three

different isoforms because of alternative splicing (Love et al., 2003, Hanover et al., 2003). The

main difference between isoforms is the number of tetratricopeptide repeats in N-terminal

region. The OGT isoforms differ also in their tissue distribution and probably they may have

different functions (Lazarus et al., 2006).The cloned sequence of O-GlcNAcase was found to be identical to that of MGEA5(meningioma-expressed antigen 5), which was identified genetically in human meningiomas(Heckel et al., 1998; Comtesse et al., 2001).MGEA5 localizes to chromosome 10q24.1-q24.3region. O-GlcNAcase seems to be a bifunctional enzyme. The N-terminus containsO-GlcNAc hydrolase activity and C-terminus bears a putative histone acetyltransferase(HAT) domain (Toleman et. al, 2004). O-GlcNAcase is reported both HAT and O-GlcNAcaseactivity in vitro.

There is growing evidence that perturbation in O-GlcNAc signaling is involved in thepathology of cancers. Overexpression of OGT causes defective cytokinesis increasing

polyploidy of cells, a feature common to many cancer cells (Slawson et al., 2005). O-GlcNAcis present on many transcription and cell cycle regulatory proteins. The protooncogenec-Myc which regulates transcription of genes involved in cell proliferation, apoptosis andmetabolism is modified by O-GlcNAc at Thr58 and this potentially stabilizes the protein(Kamemura et al., 2002). The important tumor suppressor p53, which is mutant ordysregulated in many cancers bears O-GlcNAc at Ser149. Increased O-GlcNAcylation of p53at Ser149 results in decreased p53 ubiquitination and stabilizes the p53 protein (Yang WH etal., 2006).

Several malignancies have been shown to have increased level of O-GlcNAcylationcompared to normal tissue (Gu Y et al., 2010; Mi et al., 2011; Caldwell et al., 2010). Elevated

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OGT mRNA expression was found in breast cancer cell lines and breast invasive ductalcarcinoma compared with normal breast cells and normal breast tissue (Caldwell et. al.,2010; Krzelak et al., 2011a). It has been shown that poorly differentiated breast tumors(grade II and III) had significantly higher OGT expression than grade I tumors and lymph

node metastasis is significantly associated with decreased MGEA5 mRNA expression(Krzelak et al., 2011a). The intracellular O-GlcNAcylation has been found to be alsoassociated with the pathogenesis of chronic lymphatic leukemia CLL (Shi et al., 2010).

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abundant in non-neoplastic lesions than in tumors (Krzelak et al., 2010). However, ourpreliminary studies concerning O-GlcNAc cycling enzymes expression did not show anysignificant differences in mRNA expression of MGEA5 between thyroid cancers and non-neoplastic lesions (Fig.3). Contrary, OGT mRNA expression seems to be elevated in cancer

samples in comparison to nodular goiters or benign tumors. Moreover, expression of OGTmRNA was higher in papillary carcinoma with lymph node metastasis compared to non-metastatic carcinoma. High expression was also found in case of anaplastic carcinoma.These results might suggest that O-GlcNAc transferase is associated with thyroid cancerprogression.

Caldwell et al. (2010) have provided evidence that the reduction abnormally elevated OGTand O-GlcNAc levels in breast cancer cells inhibits cancer cell growth in vitro and in vivo andalso diminishes breast cancer invasion. Decreasing O-GlcNAc levels through knockdown ofOGT in cancer cells promotes elevation of the cell-cycle regulator p27kip1 and reducesexpression of FoxM1 and its transcriptional target matrix metalloproteinase-2. Our studies

concerning O-GlcNAc role in anaplastic thyroid cancer cells 8305C showed that down-regulation of O-GlcNAcase enhanced both basal and IGF-1 stimulated Akt1 activation andcell proliferation (Krzelak et al., 2011b). In cells treated with O-GlcNAcase inhibitor

PUGNAc or specific for O-GlcNAcase siRNAs phosphorylation of kinase GSK3 and cyclinD1 level were higher than in control cells. These findings suggest that increasedproliferation of 8305C cells with down-regulation ofO-GlcNAcase at least partially depends

on IGF-1 Akt1 GSK3 cyclin D1 pathway.

Although abnormal O-GlcNAcylation seems to be a feature of cancer cells its role intumorigenesis and cancer progression has not been fully elucidated and furtherinvestigations in this area are needed. However, it seems that O-GlcNAc transferase

expression might be a marker for some tumor progression including thyroid. Moreover,regulation of O-GlcNAc may be a promissory novel therapeutic strategy for cancer and OGTmay be in future a potential drug target.

3. Glycoproteins as thyroid cancer biomarkers

Glycoproteins seem to provide an abounding source for discovering biomarkers for thyroidlesions. Gene expression profiling studies identified some genes coding glycoproteins forexample MET, SERPINA1, TIMP1, FN1, CD44, SDC4, DPP4 and PROS1 as importantthyroid cancer markers (Griffith et al., 2006). These significantly deregulated genes inthyroid cancer cells may help to develop a panel of markers with sufficient sensitivity and

specificity for the diagnostic purpose. Studies of cell surface and secreted protein profiles ofhuman thyroid cancer cell lines (FTC-133, TPC-1, XTC-1, ARO, DRO-1) revealed distinctglycoprotein patterns for each cell line (Arcinas et al., 2009). Of the 333 glycoproteinsidentified in the five thyroid cancer lines, nearly one third, 105, were found exclusively in asingle cell line. FTC-133 and ARO had the fewest (7) and the most (34) uniquely identifiedglycoproteins, respectively. It is suggested that several glycoproteins have a potential asbiomarkers for a specific type of thyroid cancer or as general thyroid cancer biomarkers. Forexample NCAM-1 is a glycoprotein biomarker candidate for differentiated cancers,syndecan-1 and cadherin-13 for follicular carcinoma, elastin microfibril interfacer-1,hyaluronan and proteoglycan link protein 1, ephrin-B-1 for anaplastic carcinoma, CD157 forHrthle cell thyroid carcinoma (Arcinas et al., 2009).

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Usefulness of some glycoprotein expression analysis for thyroid cancer diagnostics havebeen already extensively tested.

3.1 Thyroglobulin

Thyroglobulin (Tg) is a large glycoprotein produced by thyroid normal follicular cells anddifferentiated malignant cells. The production of Tg is low in poorly differentiated cells andabsent in anaplastic thyroid cancer (Francis & Schlumberger, 2008). Thyroglobulin providesa matrix for the synthesis of the thyroid hormones, thyroxine (T4) and triiodothyronine (T3),and acts as a storage vehicle for iodine, in the form of iodinated tyrosyl residues (Vali et al.,2000). Tg is a homodimer with molecular weight of 660 kDa and contains 10% ofcarbohydrate structures. Of the 20 putative N-linked glycosylation sites in the humanthyroglobulin polypeptide chain, 16 were shown to be actually glycosylated in the matureprotein (Yang SX et al., 1996). Eight of these confirmed glycosylation sites appear to belinked to complex-type oligosaccharide units containing fucose and galactose in addition to

mannose and glucosamine. Five sites contain high mannose type units and two sites arelinked to oligosaccharide units that may be either hybrid or complex structures (Yang SX etal., 1996). The oligosaccharides of Tg have been shown to affect the structure and function ofTg. They have an impact iodination and hormone synthesis, targeting of Tg to subcellularand extracellular compartments, interactions with a putative membrane receptor andimmunoreactivity of Tg (Vali et al., 2000).

Owing to thyroglobulin tissue specificity, its serum level is widely used as a marker forrecurrence of thyroid carcinoma following total thyroidectomy. The improved sensitivity ofthyroglobulin assays and the adoption of an international calibration standard have madethyroglobulin measurement an essential part of thyroid cancer monitoring (Lin, 2008; Ringel

& Ladenson 2004). However, it ought to be remembered that there are some limitations ofserum thyroglobulin measurement. The presence of anti-thyroglobulin antibodies interfereswith measurements of Tg protein made either by immunometric assay or byradioimmunoassay (Feldt-Rasmussen & Rasmussen, 1985; Mariotti et al, 1995; Spencer,2004). Due to difficulties with antibody interference, there has been an interest in developingnew techniques for thyroid carcinoma monitoring. One of these techniques is the detectionof circulating thyroid cells by the measurement of thyroglobulin mRNA (Tg-mRNA) inperipheral blood (Ditkoff et al., 1996). However, there are contradicting results on theusefulness of Tg-mRNA detection in peripheral blood. Some authors suggested theusefulness of this method in the follow-up of thyroid cancer patients (Ringel et al., 1998,1999; Biscolla et al, 2000; Savagner et al., 2002) but others questioned its reliability (Bojunga

et al., 2000; Eszlinger et al., 2002; Span et al., 2003, Elisei et al., 2004). Thyroglobulin mRNAmay be expressed in very low concentrations by other cells in the body, including peripherallymphocytes, through a process known as illegitimate transcription (Verburg et al., 2004).

Since the serum Tg level is increased in the majority of patients with either benign ormalignant thyroid nodules, its use is not recommended in the preoperative differentiationbetween thyroid lesions. However, it is suggested that the carbohydrate structure analysis ofthyroglobulin might be useful for thyroid cancer diagnostics. During malignanttransformation, epithelial cells modulate the glycosylation profile of their secretion proteinsand these post-translational changes may represent specific markers which may be usefuldiagnostic or prognostic tools. The composition of carbohydrate chains on thyroglobulin

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from thyroid carcinoma has been reported to differ from that in normal thyroid tissue(Maruyama et al., 1998; Shimizu et al., 2007a). Shimizu et al. (2007b) investigated Lensculinaris agglutinin (LCA)-reactive thyroglobulin ratio in serum to evaluate its usefulness fordistinguishing between thyroid carcinoma and benign thyroid tumor. Their results were

very promising. The Lens culinaris agglutinin-reactive thyroglobulin ratio was significantlylower in patients with thyroid carcinoma than in patients with benign thyroid tumor.Moreover, in cases of thyroid carcinoma with lymph node metastasis, Lens culinarisagglutinin-reactive Tg ratios were significantly decreased compared to patients with thyroidcarcinoma without metastasis. Further studies confirm usefulness of the determination ofLCA-reactive Tg ratios using an enzyme-linked immunosorbent assay for distinguishingbetween benign and malignant lesions (Kanai et al., 2009).

3.2 Fibronectin

Fibronectin (FN) is a high molecular mass adhesive glycoprotein present in the extracellular

matrix that plays a significant role in cell adhesion, migration, growth, differentiation andthe maintenance of normal cell morphology (Pankov & Yamada, 2002). Although fibronectinis encoded by a single gene it can exist in 20 different isoforms in humans as a results ofalternative splicing and post-translational modifications. Fibronectin usually exists as adimer composed of two ~250 kDa subunits linked by a pair of disulfide bonds near the C-termini. Subunits are composed primarily of three types of repeating module (I, II and III).Sets of modules make up domains for binding to variety of extracellular and cell surfacemolecules including collagen, glycosaminoglycans, fibrin, integrins and FN itself(Wierzbicka-Patynowski & Schwarzbauer, 2003). FN isoforms can be divided in two groups:soluble plasma FNs which are synthesized predominantly in the liver by hepatocytes andthe less-soluble cellular FNs synthesized by other cell types. Heterogenity of fibronectin isalso caused by post-translational modification. FN isoforms are glycoproteins that contain

4-9% carbohydrate, depending on the cell type. Glycosylation sites reside predominantlywithin type III repeats and the collagen-binding domain. The physiological role of thecarbohydrates is not certain, but they appear to stabilize FN against hydrolysis andmodulate its affinity to some substrates (Pankov & Yamada, 2002). There are seven potentialN-glycosylation sites in human FN, and these N-glycans are largely responsible for thecarbohydrate content of this molecule. The N-glycan of plasma FN is composed of complex-type biantennary oligosaccharides and is largely sialylated, whereas fibroblast-derivedcellular FN contains fucose linked to the innermost N-acetylglucosamine (GlcNAc) and isless sialylated (Fukuda et al., 1982, Tajiri et al., 2005). Fetal and neoplastic cells

predominantly express a uniquely glycosylated isoform called oncofetal fibronectin(OnfFN). This isoform is characterized by the presence of oncofetal domain situated in the

COOH terminal region, which is absent in normal fibronectin. The oncofetal domain of FNis composed of an oligosaccharide linked to a hexapeptide by O-glycosylation (Matsuura etal., 1988).

Fibronectin 1 was first reported to be overexpressed in papillary carcinomas (Huang et al.,2001; Wasenius et al., 2003). Prasad et al. (2005) found that FN1 expression was significantlyassociated with malignancy and highly specific for carcinomas compared to adenomas andnon-neoplastic tissues. Immunohistochemical analysis showed that FN1 was expressed in 61of 67 papillary carcinoma cases, 3 of 6 follicular carcinoma, 4 of 4 anaplastic carcinoma and

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6 of 8 Hrthle cells carcinoma cases. Contrary expression of FN1 was observed only in 2 of29 samples of nodular goiters an 1 of 21 adenoma cases. Coexpression of FN1 with othermarkers such as galectin-3 or HBME1 was observed only in carcinomas (100% specific)while concurrent absence of FN1 and galectin-3 or HBME1 (Fn_Gal-3_ or FN_HBME1_

immunophenotype) was highly specific (96%) for adenomas (Prasad et al., 2005). Thus, thisstudy suggest that fibronectin 1 is a very useful marker in the diagnosis of thyroidcarcinomas. However, some other studies did not confirm high utility of fibronectin 1.Immunohistochemical studies of Nasr et al. (2006) showed that although FN1 was quitespecific for papillary carcinomas, a large portion of PTC (31%) were negative. Even thosecases that were positive showed weak and focal staining, and there was often significantbackground staining. The authors suggested that low sensitivity, focal staining and highbackground positivity significantly impair the utility of FN1 in diagnostics.

Using the reverse transcription-PCR technique, Takano et al. (1998, 1999) have suggestedthat oncofetal fibronectin (onfFN) may be an accurate molecular preoperative diagnostic

marker for papillary thyroid carcinoma. They analyzed the expression of onfFN in fine-needle aspiration biopsies. The sensitivity and specificity of this method were 96% and100%, respectively. They also showed high onfFN expression in anaplastic cancer tissuesand cell lines (Takano et al., 2007). It has been suggested that oncofetal fibronectin may be abetter tumor-specific marker in detecting minimal residual disease in differentiated thyroidcarcinoma than thyroglobulin (Hesse et al., 2005). Thyroglobulin mRNA was thought tooriginate from circulating thyrocytes, but other studies demonstrated that physiologicalblood cells such as leukocytes are capable of ectopic Tg mRNA transcription (Verburg et al.,2004) In order to monitor patients with DTC for minimal residual disease by blood assays,Hesse et al. (2005) developed and optimized a specific, sensitive real-time RTPCR assayusing FRET technology to quantify absolute amounts of onfFN templates. High expressionrate of onfFN transcripts in DTCs was demonstrated, while onfFN mRNA was not found tobe illegitimately transcribed by peripheral blood cells. The authors suggested that onfFNmRNA analysis may be a specific tool for monitoring micrometastases in the context ofminimal residual disease or for assessing tumor response to therapy. Continuing theirstudies they performed the analysis of mRNA level in peripheral blood of PTC patients who

were previously treated by thyreoidectomy and treated by levothyroxine to determine ifonfFN levels are correlated with the status of the disease or with thyroid-stimulatinghormone (TSH) serum concentrations (Wehmeier et al 2010). The mean value of onfFNmRNA in bloods from healthy subjects was used as control. OnfFN transcripts were highlyabundant in the peripheral blood of the patients, but the levels of onfFN mRNA did not

differ significantly among the patients who were free of the disease, had local residualdisease or metastatic disease. However, there was a trend towards higher expression rates ofonfFN mRNA in patients with metastases than those free of disease. Discrimination betweenthe disease states was better without TSH stimulation. The authors suggest that circulatingonfFN mRNA, may be a useful tool to detect circulating thyroid cancer cells.

3.3 CD44

The CD44 glycoprotein is an acidic molecule whose charge is largely determined by sialicacid. CD44 plays a critical role in a variety of cellular behaviors, including adhesion,migration, invasion, and survival. The main ligand for CD44 is a hyaluronan but CD44 has

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an afifinity to other extracellular matrix constituents such as osteopontin, collagens, andmatrix metalloproteinases (MMPs) (Cichy & Pur, 2003). The gene for CD44 contains 20exons, 12 of which are expressed by the most common form of CD44, referred to as standardCD44 (CD44s). Isoforms of CD44 are generated by the insertion of alternative exons (V1

V11) at a single site within the membrane-proximal portion of the extracellular domain(Ponta et al., 2003). Molecules containing the variable exons or their peptide products aredesignated CD44v. The most abundant standard protein consists of three regions, a 72amino acid (aa) C-terminal cytoplasmic domain, a 21 aa transmembrane domain, and a 270aa extracellular domain (Goodison et al., 1999). The degree of glycosylation can affect theligand binding characteristics of the protein and therefore alter its function. The regulationof the amount and the type of post-translational modification can add further diversity tothe range of potential functions of CD44 isoforms. Expression of multiple CD44 isoforms isfrequently upregulated during neoplastic transformation. CD44, particularly its variants,may be useful as a diagnostic or prognostic marker of malignancy (Goodison et al., 1999).

Expression of CD44 both on mRNA and protein levels was intensively studied in thyroidpathological lesions in order to evaluate its utility in distinguishing benign and malignanttumors. Immunohistochemical and immunocytochemical analyses showed that CD44molecule was preferentially expressed in papillary thyroid carcinoma compared with otherthyroid neoplasms and non-neoplastic lesions (Figge et al., 1994; Bhm et al., 2000; Kim etal., 2002). Variant isoforms of CD44 (CD44v3 and CD44v6) were detected in follicular andpapillary carcinomas as well as in follicular adenomas but not in non-neoplastic lesions (Guet al., 1998). The frequency of both variants expression was significantly higher in PTC withnode metastasis than in PTC without metastasis (Gu et al., 1998). Since CD44v6 is notexpressed by normal thyrocytes and is variably detected in benign and malignant lesionsGasbarri et al. (1999) suggested that it could be a preoperative marker to identify malignant

lesions. Its immunodetection in cytologic specimens obtained by fine-needle aspirationbiopsy could be useful for selecting those nodular lesions of the thyroid gland that need tobe surgically resected (Gasbarri et al., 1999). Bhm et al. (2000) found that reduced level ofCD44s in differentiated thyroid cancer patients seemed to be an independent prognosticfactor for unfavorable disease outcome. Age older than 60 years, distant metastases andadvanced pTNM stage were related to the loss of CD44s expression.

The main problem in preoperative diagnostics of thyroid neoplastic lesions is distinguishingbetween follicular carcinomas and follicular adenomas. Nasir et al. (2004) tested usefulnessof CD44v6 among other markers in differentiating benign and malignant follicularneoplasms. They found membranous CD44v6 staining in 81% of FTC but in only 20% of

follicular adenomas and suggested that its immunohistochemical detection may be usefulfor follicular neoplasm diagnostics. Maruta et al. (2004) analysed CD44v6 immunostainingin fine-needle aspiration cytology of 35 follicular carcinoma specimens and 44 cases offollicular adenomas and observed positive reaction in 74% of carcinoma cases and 30% ofadenomas. There was no correlation between the expression of CD44v6 in follicular

carcinomas and characteristics such as capsular invasion, vascular invasion, metastasis, ortumor size (Maruta et al., 2004).

Since CD44 is often detected in benign lesions its diagnostic importance is questioned bysome authors (Nikiel et al., 2006). Matesa et al. (2007) analyzed CD44v6 mRNA expressionin order to answer whether the presence of macrophages and Hrthle cells are responsible

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for positive expression detected in benign lesions. They found a statistically significantrelationship between presence of Hrthle cells and positive expression of CD44v6 innodular goiter cytological samples.

3.4 Osteopontin

Osteopontin (OPN) is an acidic hydrophilic glycophosphoprotein rich in aspartic acid,glutamic acid and serine and contains ~30 monosaccharides, including 10 residues of sialicacid. One carbohydrate chain is attached by N-glycosyl bond to protein and 5-6 chains arelinked by O-glycosyl bond (Rangaswami et al., 2006). OPN is a member of a small integrinbinding ligand N-linked glycoprotein (SIBLING) family of proteins which include bonesialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialoprotein (DSPP), and matrixextracellular phoshoglycoprotein (MEPE) (Wai & Kuo, 2008). OPN functions by mediatingcell-matrix interactions and cellular signaling through binding with integrin and CD44receptors. Many studies have indicated that OPN is highly expressed in several

malignancies. OPN expression is associated with tumor invasion, progression or metastasisin breast, stomach, lung, prostate, liver and colon cancers (for a review see Wai & Kuo, 2008;Rangaswami et al., 2006).

Castellone et al. (2004) showed that osteopontin was a major RET/PTC - inducedtranscriptional target in PC Cl3 thyroid follicular cells. RET/PTC also induced a strong

overexpression of CD44 which is a cell surface signaling receptor for OPN. These resultsprompt further studies to ascertain whether OPN could be a useful PTC tumor marker.

OPN expression was studied by immunohistochemistry in 117 samples of thyroid papillary

cancer, benign lesions and normal tissues (Guarino et al., 2005). OPN was found to be

overexpressed in PTCs compared with normal thyroid tissue, follicular adenomas and

multinodular goiters. Moreover, OPN up-regulation was correlated with aggressiveclinicopathological features of PTC. The prevalence and intensity of OPN staining were

significantly correlated with the presence of lymph node metastases and tumor size.However, follicular variant of PTC had lower prevalance of OPN expression compared to

classical type. Studies of Briese et al. (2010) on a large number of thyroid samples showed

that normal thyroid was negative for OPN, thyroid adenomas were weakly OPN positive,whereas many carcinomas were strongly positive. However, there was no association

between OPN expression and tumor size and metastasis status.

mRNA level of SPP1 (osteopontin gene) was validated in a panel of 57 thyroid tumorsusing quantitative PCR (qPCR) (Oler et al., 2008). SPP1 was overexpressed in PTCs but the

difference was not considered significant. However, SPP1 expression was associated withthe presence of lymph node metastasis for tumors >1 cm. The expression levels of SPP1 infollicular variant of PTC and follicular carcinoma were lower than in classical type ofPTC.

Recently Wang et al. (2010) found that osteopontin expression is positively correlated inthyroid papillary carcinoma with phosphorylated c-Jun kinase (p-JNK). Activation of theJNK signaling pathway appears to be an important event in thyroid tumorigenesis and,perhaps, in tumor progression making p-JNK a possible target in cancer treatment.Coordinated expression of p-JNK and OPN immunoreaction suggest an involvement ofboth genes in the same molecular pathway in thyroid lesions (Wang et al., 2010).

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3.5 NCAM

Neuronal cell adhesion molecule (NCAM, CD56) mediates homotypic and heterotypic cell-cell adhesion (Crossin & Krushel, 2000). NCAM structurally belongs to the immunoglobulin

superfamily. The extracellular part of NCAM consists of five Ig-like domains and twofibronectin type III-like domains. Alternative mRNA splicing results in three majorisoforms: a 120 kDa isoform which is predominantly expressed in normal and welldifferentiated tissues and a 140 and 180 kDa isoforms that are found predominantly in lessdifferentiated or malignant cell types (Jensen & Berthold, 2007). NCAM is unique amongadhesion molecules because it carries a large amount of the negatively charged sugar,polysialic acid (PSA) (Crossin & Krushel, 2000). The presence of PSA can affect the strengthor stability of adhesion systems in which NCAM is involved.

NCAM is present on follicular epithelial cells of the normal thyroid but its expression is

reduced by malignant transformation and may affect the migratory capability of tumor cells

(Zeromski et al., 1998, 1999; Satoh et al., 2001). Scarpino et al. (2007) analyzed NCAMexpression in tissue sections of 61 cases of papillary thyroid carcinoma (PTC) using

immunohistochemistry and quantitative real-time PCR. Reduced NCAM protein expressionwas observed by immunohistochemistry in all histological variants of PTC and also in

lymph node metastases. Reduced expression of NCAM protein was associated with a

significant reduction of NCAM mRNA in the tumor tissue compared with the paired

normal thyroid tissue. Other studies showed NCAM to be extremely useful in the

distinction between PTC and follicular benign or malignant lesions (El Demellawy et al.,2008, 2009; Ozolins et al., 2010). El Demellawy et al. (2009) evaluated the diagnostic value of

protein expression using antibodies against NCAM in normal follicular thyroid epithelium,benign thyroid lesions, and thyroid carcinomas. Their aim was to study the applicability of

difference in NCAM expression as a marker that distinguishes PTC, including the follicularvariant from other follicular thyroid lesions and follicular thyroid carcinoma. They found

that NCAM is of value in distinguishing PTC from other thyroid follicular pathology with a

sensitivity of 100% and a specificity of 100%.

The potential role of NCAM in tumor cell biology was investigated by silencing the NCAM

gene in the TPC1 thyroid papillary carcinoma cell line (Scarpino et al., 2007). The resultsconfirm that modifications of NCAM expression cause profound alterations in the adhesiveand migratory properties. However, contrary to the observation that loss of NCAM is

usually associated with increased tumor invasiveness in vivo, NCAM-silenced TPC-1 cellswere more adhesive to different extracellular matrix components, and were less efficient in

cell migration and invasiveness. This discrepancy requires further investigations.

3.6 TIMPs

Tissue inhibitors of metalloproteinases (TIMPs) have a dual role in the process of tumorprogression with both pro- and anti-tumorigenic activities. Although originallycharacterized by their ability to inhibit matrix metalloproteinases (MMPs) activity, TIMPshave additional biological activities and have been shown to regulate a number of cellularprocesses including cell growth, migration, and apoptosis (Stetler-Stevenson, 2008 ). FourTIMPs have been currently characterized in human and designated as TIMP-1, -2, -3 and -4.They are expressed by a variety of cell types and are present in most tissues and body fluids.

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TIMP-1 genediffers from the other members of the family in having a short exon 1 that istranscribed but not translated. The function of exon 1 appears to be related to the control ofthe specificity of tissue expression and may contain tissue-specific repressor elements.TIMP-1 is a glycosylated protein and the N-linked oligosaccharides are composed of sialic

acid, mannose, galactose and N-acetylglucosamine. The TIMP-1 glycosylation seems to playa role in various functions including correct folding of the nascent protein, transport of themolecule to cell surface and enhanced stability of the protein (Lambert et al., 2004). TIMP-3has also one glycosylation site.

TIMP-1 mRNA or protein overexpression was found in thyroid papillary cancers compared tonormal tissue or goiters (Hawthorn et al., 2004; Maeta et al., 2001). Shi et al. (1999) investigatedTIMP-1 gene expression in 39 primary thyroid tumors to see whether there is a correlationbetween TIMP-1 expression and the aggressiveness of the disease. They also transfectedhuman TIMP-1 cDNA into a papillary thyroid carcinoma cell line (NPA) to study the effect ofTIMP-1 expression on its invasive potential using an in vitro tumor invasion assay. The results

showed that TIMP-1 mRNA level correlated directly with tumor aggressiveness: the highestnumber of TIMP-1 transcripts was found in stages III and IV versus benign goitres. However,overexpression of TIMP-1 by gene transfer resulted in a significant suppression of themalignant phenotype of NPA cells which suggests that TIMP-1 may function as a thyroidtumor invasion/metastasis suppressor. To explain these discrepancies, the authors suggestedthat the increased levels of TIMP-1 transcripts observed in cancer samples came from stromacells to counteract tumor invasion and metastasis.

Kebebew et al. (2006) performed a real-time quantitative reverse-transcriptase polymerasechain reaction (RT-PCR) assay of several candidate diagnostic markers to distinguish benignfrom malignant thyroid neoplasms, and to predict the extent of the disease. TIMP-1 mRNAexpression level as well as ECM1 (extracellular matrix protein 1), TMPRSS4 (transmembrane

protease, serine 4) and ANGPT2 (angiopoietin 2) mRNA levels were found to beindependent diagnostic markers of malignant thyroid neoplasms. The AUC (the area underthe receiver operating characteristic (ROC) curve) for 4 diagnostic genes in combinationwas 0.993 with sensitivity of 100%, specificity of 94.6%, positive predictive value of 96.5%,and negative predictive value of 100%. Thus the RT-PCR multigene assay involving TIMP1is an excellent diagnostic marker for differentiated thyroid cancer and it will be a helpfuladjunct to FNA biopsy of thyroid nodules.

The plasma concentration of MMP-1, 2, 3, 8 and 9 as well TIMP-1 and 2 was evaluated byenzyme-linked immunosorbent assay (ELISA) in patients with thyroid cancers (papillary,anaplastic and medullary) and in healthy subjects. The author suggests that, predominanceof MMP-2 over TIMP-2 and TIMP-1 over MMP-1 is a common feature in patients withthyroid cancer (Komorowski et al., 2002).

Recently Nam et al. (2011) have studied the expression of matrix metalloproteinase-13(MMP-13) and tissue inhibitor of metalloproteinase-13 (TIMP-3) in thyroid cancer by RT-PCR. They found that MMP-13 and TIMP-3 expression levels were significantly decreased inPTC samples compared with normal thyroid tissues.

3.7 Dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPPIV), assigned to the CD26 cluster, is a multifunctional proteinexpressed on epithelial cells and lymphocytes. DPPIV is a type II transmembrane

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glycoprotein which consists of a large extracellular domain (C-terminus) connected by aflexible stalk region to a hydrophobic transmembrane domain and short intarcellular tail (N-terminus) (Kotackov et al., 2009). It preferentially cleaves N-terminal dipeptides frompolypeptides with proline or alanine in the penultimate position and regulates the activities

of a number of hormones, neuropeptides, cytokines and chemokines (Havre et al., 2008;Kotackov et al., 2009). DPPIV palys an important role in immune regulation, signaltransduction, chemotaxis, cell adhesion and apoptosis. It is suggested that DPPIV is alsoinvolved in the neoplastic transformation and tumor progression (Pro & Dang, 2004;Kotackov et al., 2009).

Numerous early studies showed that DPPIV is abnormally expressed in thyroid carcinomasand may be useful for the diagnosis of thyroid tumors (Aratake et al., 1991; Kotani et al.,1992; Tanaka et al., 1995; Umeki et al., 1996; Tang et al., 1996; Hirai et al., 1999). DPPIV-likeenzymatic activity and DPPIV protein expression were up-regulated in thyroid cancers.However, DPPIV positivity is limited to the group of well-differentiated carcinomas,

particularly papillary carcinoma (Kholov et al., 2003; de Micco et al., 2008). DPPIVsensitivity to malignant follicular tumors including the follicular variant of PTC was lowwith a misdiagnosis rate of 2030%, especially with tumors presenting Hrthle or tall-cellfeatures. Although, the use of DPPIV can increase specificity and positive predictive valueof the cytological diagnosis, the value of the marker in the individual cases is very limited(Kholov et al., 2003). The analysis of DPPIV mRNA level in 102 PTCs and 77 normalthyroid fragments with the use of Q-PCR reaction confirmed the increase of DPPIVexpression in papillary thyroid carcinoma. However, the ROC analysis revealed that thediagnostic efficiency of DPPIV estimation is limited. Thus diagnostic usefulness of DPPIV asa single PTC marker is also doubtful (Og et al. 2006).

3.8 E-cadherin and dysadherin

Cadherins are members of a large family of transmembrane glycoproteins, many of which

participate in Ca2+-dependent homophilic cell-cell adhesion and play an important role in

the formation of tissue architecture (Shapiro & Weis, 2009). E-cadherin is necessary for

normal epithelial function. Dysadherin is a cancer-associated cell membrane glycoprotein. It

inactivates E-cadherin function in a post-transcriptional manner, has an anti-cell-cell

adhesion function and plays an important role in tumor progression and metastasis (Ino et

al., 2002).

Several authors have investigated the expression of E-cadherin in thyroid cancers

(Scheumman et al., 1995; Serini et al., 1996; von Wasielewski et al., 1997; Kapran et al., 2002;Rocha et al., 2003; Kato et al., 2002; Choi et al. 2005; Brecelj et al., 2005). In general, it appearsthat E-cadherin expression is retained in follicular neoplasms but it is reduced in papillarycarcinomas and lost in anaplastic and poorly differentiated carcinomas. Reduction or loss ofE-cadherin has been correlated with widely invasive growth and lymph node metastases(Naito et al., 2001). E-cadherin reactivity is reported to be an important prognostic factor inpapillary thyroid carcinomas (Scheumman et al., 1995; Rocha et al., 2003). Lack ofE-cadherin expression has been considered as an adverse prognostic factor for survival.

Dysadherin expression in thyroid cancer was reported by two studies. Sato et al. (2003)analyzed by immunohistochemistry dysadherin and E-cadherin expression in 92 thyroid

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carcinomas. Dysadherin was detected in 39 of 51 papillary carcinomas and in all 31undifferentiated carcinomas but not in follicular carcinomas or normal thyroid tissuecontrols. Dysadherin expression correlated significantly with tumor size, regional lymphnode metastasis, and distant metastasis of the primary carcinoma. A significant association

was also observed between dysadherin expression and death from thyroid carcinoma.Dysadherin expression showed a significant negative correlation with E-cadherinexpression (Sato et al., 2003).

Batistatou et al. (2008) compared the dysadherin expression in papillary carcinomas andpapillary microcarcinomas (PMC) to find out whether there are any differences in the cellcell adhesion system between these two malignancies that have different biologicalbehavior. PMC has been defined as a papillary neoplasm measuring 1cm or less in diameterand usually remains clinically silent and is often identified incidentally in surgicallyremoved thyroid glands for other reasons (e.g. nodular hyperplasia, thyroiditis).A statistically significant difference in dysadherin and E-cadherin expression between PC

and PMC and a negative correlation between E-cadherin and dysadherin expressionregardless of tumor size were noted (Batistatou et al., 2008).

3.9 Mucins

Mucins are defined as high molecular weight glycoproteins that contain tandem repeatstructures extensively glycosylated through GalNAc O-linkages at the threonine and serineresidues. To date, twenty one members of human mucin (MUC) family have been reportedand they are designed chronologically in order of discovery. On the basis of their structuraland physiologic characteristics, mucins have been divided into three sub-classes: thesecreted/gel-forming mucins, the soluble mucins, and the membrane- associated mucins

(Kufe, 2009; Ohashi et al., 2006).Mucins are multifaceted glycoproteins that play a crucial role in maintaining homeostasis

and promoting cell survival. They form a physical barrier, which separates an apical surfaceof epithelial cells from the external environment and protects against infection and

proteolytic degradation. The membrane-associated mucins are anchored to the surface of thecell by a transmembrane domain and then have short cytoplasmic tail that interacts with

cytoskeletal elements and cytosolic adaptor proteins. Therefore, they can act as putative

receptors that engage diverse signaling pathway linked to differentiation, proliferation and

apoptosis. Several reports indicate that aberrant upregulations of mucins especiallytransmembrane forms may promote the malignant phenotype of human carcinomas. In

many types of tumor overexpression of transmembrane mucins contribute to theoncogenesis by disrupting epithelial polarity and cell-cell interactions, to constitutiveactivation of growth and survival pathways and to blocking stress-induced apoptosis and

necrosis (Bafna et al., 2010; Kufe, 2009; Carraway et al., 2003).

The production of mucins has been relatively frequently observed in thyroid carcinomas. In astudy of 142 cases of thyroid neoplasms, Mlynek et al. (1985) found mucinous substances,which occurred in about 50% of the papillary and medullary carcinomas, 35% of the follicularcarcinomas and 21% of the anaplastic varieties. The most widely studied mucin, associatedwith thyroid malignancies is MUC1, encoded by gene located on the chromosome 1q21-24.Wreesmann et al. (2004) suggested that up-regulation of MUC1 expression in papillary thyroid

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carcinoma correlates with 1q21 amplifications and aggressive behavior. The MUC1 geneoverexpression was present in 97,5% of tall-cell variant of PTCs (TCV) as compared with only35% of conventional PTCs. These results are in contrast to the previous analysis of Biche et al.(1997) who detected no MUC1 gene amplification in 14 tumor samples (6 adenomas and 8

PTC) informative for the MUC1 gene. However, these studies showed higher MUC1expression level in 6 out of 11 papillary carcinoma cases in comparison with 10macrofollicular adenoma cases and normal thyroid tissue. Moreover, the authors observedthat intracytoplasmic MUC1 staining occurred in 75% (3/4) of high-risk papillary thyroidcarcinomas and in only 28,5% (2/7) of the low-risk PTC without extrathyroidal invasion orlymph node involvement, suggesting that MUC1 can be associated with more aggressivetumors. Indeed, comparing human thyroid cancer cell lines, Patel et al. (2005) found increasedMUC1 expression in aggressive thyroid cancer cell lines (8305C, KAT4, ARO, BHP2-7, BHP10-3, BHP7-13, BHP18-21) but not in other papillary and follicular thyroid cancer cell lines (KAT5,KAT10, NPA, WRO, FRO). In addition, they demonstrated that targeting MUC1 withmonoclonal antibody selectively affects cell viability and confirmed that MUC1 plays a crucialrole in the thyroid behavior. Furthermore, a relationship was observed between cytoplasmicMUC1 and cyclin D1 immunostaining in the conventional PTCs and in papillarymicrocarcinomas (PMCs), implying that MUC1 may be involved in the regulation of Wntpathway and has a role in B-catenin/cyclin D1 signaling. However, due to a very broad MUC1expression in different histological variants, the authors were not able to determinate itsprognostic utility (Abrosimov et al., 2007). Magro et al. (2003) found that MUC1 may co-existin variety of glycosylated forms in different cellular compartments. They supposed that post-translational modification of mucins, rather than alterations in expression levels, may bestronger associated with tumor progression and specific glycosylation traits of MUC1 andcould be an ancillary tool in histopathological diagnosis. Other investigators found MUC1

mRNA overexpression in 15 cases of PTC in contrast to 22-follicular adenomas and 22-normalthyroid tissues (Baek et al., 2007). More recently, Morari et al. (2010) basing on the study of 410patients reported that MUC1 expression distinguished benign from malignant thyroid tissuewith sensitivity of 89%; specificity of 52%; predictive positive value=75%; predictive negativevalue=74%. In conclusion, they suggested that MUC1 is not a reliable prognostic marker butmay be useful in characterization of thyroid carcinomas, especially the follicular patternedthyroid lesions. The expression of other mucins in thyroid neoplasms has been poorlyinvestigated. Nam et al. (2011) have suggested that MUC4 and MUC15 are the other importantmucins associated with thyroid transformation. They showed significantly increased MUC4and MUC15 mRNA level and positive immunoreactivities in PTC compared with normaltissue. High MUC4 expression correlated with small tumor size and papillary thyroid

microcarcinoma subtype whereas overexpression of MUC15 was associated with age, thepresence of multifocality and distant metastasis. The results of this study suggest, that MUC4and MUC15 may play a crucial role in thyroid malignancy, especially the MUC15 may be anew prognostic marker and potential therapeutic target. However, there are somediscrepancies between the previously published reports that indicated weakly positive ornegative MUC4 staining of both benign and malignant thyroid carcinomas (Baek et al., 2007;Magro et al., 2003). The expression of MUC2, MUC3, MUC5AC, MUC5B and MUC6 in thyroidneoplasms has been tested, though none of these glycoproteins was associated withclinicopathological features of thyroid carcinomas (Magro et al., 2003; Alves et al., 1999). Theclinical and prognostic significance of mucins in thyroid tumors is still an open question.

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4. Galectins

Galectins are endogenous lectins defined by shared consensus amino acid sequences and an

affinity for -galactose containing oligosaccharides. Fifteen members of galectin family have

been identified and classified into three subgroups based on their structure: prototype(galectin-1, 2, 5, 10, 11, 13, 14, 15 ), tandem repeat type (galectin-6, 8, 9, 12) and chimera type

(galectin-3). The presence of at least one carbohydrate recognition domain (CRD) is a

common characteristic of galectins (Krzelak & Lipiska, 2004; Elola et al., 2007; Boscher et

al., 2011). Galectins have both intra- and extracellular localization. Galectins can be secreted

by non-classical pathway and depending on the cell type or differentiation state, they are

found in the cytoplasm and the nucleus, on the cell surface and in the extracellular matrix

(Hughes, 2001; Haudek et al., 2010; Garner & Baum, 2008). Galectins have been shown to

play roles in diverse biological processes, such as embryogenesis, adhesion and proliferation

of cells, apoptosis, mRNA splicing and modulation of immune response (Krzelak &

Lipiska, 2004; Elola et al., 2007; Boscher et al., 2011).

Among various galectins, galectin-1, 3 and 7 have gained attention as potential markers of

thyroid malignancy. However, the data concerning galectin -1 and 7 expression in thyroid

are very limited. Chiariotti et al. (1995) analyzed mRNA and protein levels of galectin-1 in

74 human thyroid specimens of neoplastic, hyperproliferative and normal tissues. Galectin-1

mRNA levels was increased in 28 of 40 papillary carcinomas and in 6 of 7 anaplastic

carcinomas compared with normal or hyperplastic thyroid. Immunohistochemical analysis

of normal thyroid and papillary carcinoma sections revealed a higher content of galectin-1

protein in neoplastic cells than in normal cells. Similarly Xu et al. (1995) showed that all

studied by them thyroid malignancies of epithelial origin (i.e., papillary and follicular

carcinomas) and metastatic lymph node from a papillary carcinoma expressed high levels ofgalectin-1. In contrast neither benign thyroid adenomas nor adjacent normal thyroid tissue

expressed galectin-1. There are only two studies concerning galectin-7 expression in thyroid

cancers (Rorive et al., 2002; Than et al., 2008). The profile of galectin-7 expression is different

from that of galectin-1 and 3. The results of immunohistochemical analysis revealed that

this galectin is markedly down-regulated in adenomas compared with multinodular goiters

and carcinomas (Rorive et al., 2002). Than et al. (2008) did not show any diagnostic value of

galectin-7 in thyroid malignancy, even for a simple differentiation between obvious benign

and malignant thyroid lesions .

In contrast to galectin-1 and 7, galectin-3 is one of the best studied molecular markers for

thyroid diagnosis (for rewiew see Sanabria et al., 2007; Chiu et al., 2010) . The expressionof this protein was widely studied both in cancer tissues and in cytological specimens.

Numerous studies have reported that galectin-3 is a very sensitive and reliable diagnostic

marker for identification of thyroid carcinomas with high sensitivity and specificity

(Orlandi et al., 1998; Gasbarri et al., 1999; Inohara et al., 1999; Bartolazzi et al., 2001;

Saggiorato et al., 2001,2004; Pisani et al., 2004). However there are studies which did not

confirm this diagnostic utility of galectin-3 (Niedziela et al., 2002, Mehrotra et al., 2004,

Mills et al., 2005) The discrepancies observed in these reports could have resulted from

different methods used for galectin-3 expression analysis. These methods differ in sample

preparation details, the used antibody, dilution of antibodies, assessement of positive

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results (percentage of marked cells and intensity of staining). Moreover, in the thyroid

gland, oxyphilic cells are rich in endogenous biotin and galectin-3 immunocytochemistry

may provide false-positive results in oxyphilic cell lesions due to biotin-based detection

systems (Volante et al., 2004).

Recently, the diagnostic utility of galectin-3 in distinguishing benign from malignantfollicular thyroid nodules was evaluated in a multicenter trial comprised a large cohort ofpatients (Bartolazzi et al., 2008). Galectin-3 expression analysis was applied preoperativelyon 465 follicular thyroid FNAB samples and diagnostic accuracy was compared with finalhistology. Galectin-3 expression had an overall reported sensitivity of 78%, specificity of93% and accuracy of 88% for distinguishing benign and malignant thyroid tumors. Similarresults were obtained in two center trial reported by Franco et al. (2009). They comparedgalectin-3 staining results in suspicious or indeterminate FNABs samples with the histologicdiagnosis of the thyroidectomy specimens. Galectin-3 expression was found to havesensitivity of 83%, specificity of 81%, positive predictive value of 84%, and negative

predictive value of 80%.

Few studies have evaluated the correlation between galectin-3 expression and

clinicopathological parameters of thyroid cancers such as tumor size and grade, capsular

invasion or lymph node metastasis status. The galectin-3 expression level in follicular

carcinoma was significantly increased with the degree of vascular or capsular invasion (Ito

Y et al. 2005). High galectin-3 expression was also associated with lymph node metastasis in

MTC (Faggiano et al., 2002; Cvejic et al., 2005). Contrary, Kawachi et al. (2000) reported

significantly higher galectin-3 expression in the primary foci of the thyroid tumors with

lymph node metastases, but they found lower levels of galectin-3 in their metasatsis

offspring. Recently, Trkz et al. (2008) suggested that galectin-3 overexpression is more

profound in early stages of papillary carcinoma, and its expression intensity decreasesduring tumor progression.

Two studies have analyzed the serum level of galecin-3 in thyroid cancer patients (Inohara

et al. 2008; Saussez et al., 2008). They measured serum level of galectin-3 by ELISA method

and compared with histological diagnosis after thyroidectomy. Although Sauessez et al.

(2008) showed that higher serum level of galectin 3 was associated with thyroid disease

there were no significant differences between benign and malignant thyroid tissues. Inohara

et al. (2008) showed that serum galectin concentration in patients with papillary carcinomas

did not differ significantly from that in patients with follicular carcinoma or adenoma and in

healthy individuals.

Studies of Bartolazzi et al. (2008) and Franco et al. (2009) suggest that galectin-3 expression

analysis may represent useful diagnostic method for those follicular nodules that remain

indeterminate by cytological diagnosis and may improve the selection of patients for

surgery. However before it can be used in clinical practice there is an urgent need for

validation of methods of galectin-3 expression analysis. Some other studies suggest that

diagnostic usefulness of galectin-3 may be significantly increased by combining with other

potential thyroid cancer markers such as HBME1, cytokeratin-19, DPPIV, CD44v6, thyroid

peroxidase (Aratake et al., 2002; Weber et al., 2004; Maruta et al. 2004; de Matos et al., 2005;

Prasad et al., 2005; Papotti et al., 2005; Liu et al., 2008; Barut et al., 2010).

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5. Conclusion

Fine needle aspiration biopsy (FNAB) is a practical and the most effective diagnostictechnique for the preoperative detection of malignant nodule. However, the important

limitation of FNAB is the lack of sensitivity in the evaluation of follicular neoplasms due toits inability to differentiate benign follicular lesions from their malignant counterparts.Moreover, for definite diagnosis of malignancy a complete excision with unequivocalevidence of capsular or vascular invasion is required. Therefore, new markers that mayallow for accurate diagnosis of thyroid malignancy and eliminate potentially unnecessarysurgery are needed. Glycosylation changes and the analyses of expression of someglycoproteins or galectins can provide a novel strategy for improvement of thyroid cancerdiagnostics. So far, the most promising results have been obtained in the case of galectin-3expression. Currently an improved galectin-3 immunodetection method represents a newmethodological approach that can be used to optimize the preoperative selection of thyroidnodules. Further studies concerning alterations of glycosylation and glycoprotein patterns in

thyroid pathological specimens may potentially lead to the discovery of novel biomarkers.

6. Acknowledgment

This work was supported by the statutory fund for Department of Cytobiochemistry,University of Lodz.

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